1. Field of the Invention
This invention relates to immunoassays for detecting VEGF that can be used as diagnostic and prognostic methods for patients with cancer, cardiovascular, or other pathologies.
2. Description of Related Art
It is now well established that angiogenesis is implicated in the pathogenesis of a variety of disorders. These include solid tumors, intra-ocular neovascular syndromes such as proliferative retinopathies or age-related macular degeneration (AMD), rheumatoid arthritis, and psoriasis (Folkman et al J. Biol. Chem. 267:10931-10934 (1992); Klagsbrun et al Annu. Rev. Physiol. 53:217-239 (1991); and Garner A, Vascular diseases. In: Pathobiology of ocular disease. A dynamic approach Garner A, Klintworth G K, Eds. 2nd Edition (Marcel Dekker, N.Y., 1994), pp 1625-1710) In the case of solid tumors, the neovascularization allows the tumor cells to acquire a growth advantage and proliferative autonomy compared to the normal cells. Accordingly, a correlation has been observed between density of microvessels in tumor sections and patient survival in breast cancer as well as in several other tumors (Weidner et al N Engl J Med 324:1-6(1991); Horak et al. Lancet 340:1120-1124(1992); and Macchiarini et al. Lancet 340:145-146 (1992)).
The search for positive regulators of angiogenesis has yielded many candidates, including aFGF, bFGF, TGF-α, TGF-β, HGF, TNF-α, angiogenin, IL-8, etc. (Folkman et al, supra, and Klagsbrun et al., supra). The negative regulators so far identified include thrombospondin (Good et al Proc. Natl Acad. Sci. USA. 87:6624-6628 (1990)), the 16-kilodalton N-terminal fragment of prolactin (Clapp et al. Endocrinology, 133:1292-1299 (1993)), angiostatin (O'Reilly et al Cell 79:315-328 (1994)), and endostatin (O'Reilly et al. Cell 88:277-285 (1996)).
Work done over the last several years has established the key role of vascular endothelial growth factor (VEGF) in the regulation of normal and abnormal angiogenesis (Ferrara et al. Endocr. Rev. 18:4-25 (1997)). The finding that the loss of even a single VEGF allele results in embryonic lethality points to an irreplaceable role played by this factor in the development and differentiation of the vascular system (Ferrara et al., supra).
Furthermore, VEGF has been shown to be a key mediator of neovascularization associated with tumors and intra-ocular disorders (Ferrara et al, supra). The VEGF mRNA is overexpressed by the majority of human tumors examined (Berkman et al. J Clin Invest 91:153-159 (1993); Brown et al. Human Pathol. 26:86-91 (1995); Brown et al. Cancer Res. 53:4727-4735 (1993); Mattern et al Brit. J. Cancer. 73:931-934 (1996); and Dvorak et al. Am J. Pathol. 146:1029-1039 (1995)). Also, the concentration of VEGF in eye fluids is highly correlated to the presence of active proliferation of blood vessels in patients with diabetic and other ischemia-related retinopathies (Aiello et al. N. Engl. J. Med. 331:1480-1487 (1994)). Furthermore, studies have demonstrated the localization of VEGF in choroidal neovascular membranes in patients affected by acute macular degeneration (AMD) (Lopez et al. Invest. Ophtalmo. Vis. Sci. 37:855-868 (1996)).
VEGF is a heparin binding growth factor with a molecular weight of 45 kD (Plouet et al. EMBO J. 8:3801 (1989); Neufeld et al. Prog. Growth Factor Res. 5:89 (1994)). It is a dimeric glycoprotein consisting of two identical subunits. Although VEGF is encoded from a single gene, at least five isoforms exist in vivo due to alternative mRNA splicing. These isoforms, VEGF121, VEGF145, VEGF165, VEGF189, and VEGF206, contain 121, 145, 165, 189, and 206 amino acids, respectively (Leung et al. Science 246:1306 (1989); Houck et al. Mol. Endocrinol 5:1806 (1991); Tischer et al J. Biol. Chem 266:11947 (1991); Neufeld et al. The FASEB Journal 13:9-22 (1999)). The VEGF isoforms show differing affinities for heparin; VEGF121 binds heparin weakly, while VEGF165, VEGF 189, and VEGF206 bind heparin with increasing affinity. VEGF121 and VEGF165 are secreted and both isoforms are found in the circulation. In contrast, VEGF189 and VEGF206 are found mostly associated with heparin sulfate containing proteoglycans in the extracellular matrix (Houck et al. J. Biol. Chem. 267:26031 (1992); Park et al. Mol. Biol. Cell 4:1317 (1993)). Of the five isoforms, VEGF165 is the most abundantly expressed variant in the majority of cells and tissues.
Five receptors for VEGF have been identified: VEGFR-1 (FLT-1), VEGFR-2 (KDR/FLK-1), and VEGFR-3, which are all signaling tyrosine kinases, and Neuropilin-1 and Neuropilin-2, which are both accessory-isoform-specific receptors that bind selectively to VEGF165 (de Vries et al. Science 255:989 (1992); Terman et al. Biochem. Biophys Res Commun. 187:1579(1992); Millauer et al. Cell 72:835 (1993); Neufeld et al., supra). The vanous roles of these receptors in VEGF biology are under active investigation by numerous groups.
VEGF is produced by tissues and does not have to enter the circulation to exert its biological effect, but rather acts locally as a paracrine regulator. This raises the question of the significance of circulating VEGF and what role it plays in normal biology or pathology. A recent study by Yang et al. J. Pharm. Exp. Ther. 284:103 (1998) found the clearance of rhVEGF165 from the circulation to be very rapid, suggesting endogenous VEGF in the circulation is most likely the result of continual synthesis of VEGF. In addition, several studies have tried to correlate levels of circulating VEGF with tumor burden and have suggested VEGF levels as a potential prognostic marker (Ferran and Scagliotti Eur. J. Cancer 32A:2368 (1996); Gasparini et al J. Natl. Cancer Inst. 89:139 (1997); Kohn Cancer 80:2219 (1997); Baccala et al. Urology 51:327 (1998); Fujisaki et al. Am. J. Gastroenterol. 93:249 (1998)). Clearly the ability to accurately measure VEGF will be important to understand its potential role(s) in many biological processes, such as maintenance of vascular patency, menstrual cycle, ischemia, diabetes, and cancer.
The literature reports widely varying concentrations of endogenous VEGF in normal and diseased patients, ranging from undetectable to high levels. It has been reported that VEGF 165/165 can be proteolytically cleaved into three other forms: a 165/110 heterodimer, a 110/110 homodimer, and a 55-amino-acid C-terminal fragment (Keyt et al. J Biol. Chem. 271:7788-7795 (1996); Keck et al. Arch. Biochem. Biophys. 344:103-113 (1997)
The ability to measure endogenous VEGF levels depends on the availability of sensitive and specific assays. Colorimetric, chemiluminescence, and fluorometric based enzyme-linked immunosorbent assays (ELISAs) for VEGF have been reported. Houck et al., supra, (1992); Yeo et al. Clin. Chem. 38:71 (1992); Kondo et al. Biochim. Biophys. Acta 1221:211 (1994); Baker el al. Obstet. Gynecol. 86:815 (1995);Hanatani el al. Biosci. Biotechnol. Biochem. 59:1958 (1995); Leith and Michelson Cell Prolif. 28:415 (1995); Shifren et al. J. Clin Endocrinol. Metab. 81:3112 (1996); Takano et al. Cancer Res. 56:2185 (1996); Toi et al. Cancer 77:1101 (1996); Brekken et al. Cancer Res. 58:1952 (1998); Obermair et al. Br. J. Cancer 77:1870-1874 (1998); Webb et al. Sci. 94:395-404 (1998). Similar ELISAs have been successfully applied in the determination of low amounts of drugs and other antigenic components in plasma and urine samples, involve no extraction steps, and are simple to carry out.
The Houck et al., supra (1992) describe a calorimetric ELISA that appears to have ng/ml sensitivity, clearly not sensitive enough to detect endogenous VEGF levels. Yeo et al., supra (1992) describe a two-site time-resolved immunofluorometric assay, however, no VEGF was detected in normal sera (Yeo et al. Cancer Res. 53:2912 (1993)). Baker et al, supra (1995), using a modified version of this immunofluorometric assay, reported detectable levels of VEGF in plasma from pregnant women, with higher levels observed in women with preeclampsia. Similar data in pregnant women were reported by Anthony et al. Ann. Clin. Biochem 34:276 (1997) using a radioimmunoassay. Hanatani et al., supra (1995) developed a chemiluminescent ELISA capable of measuring circulating VEGF and report VEGF levels in sera from 30 normal individuals (male and female) from 8-36 pg/ml. Brekken et al, supra (1998) described ELISA assays using antibodies having binding preference to either the VEGF alone or the VEGF:Flk-1 complex.
An ELISA kit for VEGF detection is commercially available from R&D Systems (Abingdon, U.K.). The R&D VEGF ELISA kit has been used in sandwich assays wherein a monoclonal antibody is used to capture the target VEGF antigen and a polyclonal antibody is used to detect the VEGF. Webb et al. supra (1998). It is not clear whether the detection results using the R&D ELISA kit are influenced by the presence of proteolytical processes or degradation of VEGF, or by interference of other serum proteins. Obermair et al, supra (1998).
Keyt et al. J. Biol. Chem. 271:7788-7795 (1996); Keyt et al. J. Biol. Chem. 271:5638 (1996); and Shifren et al, supra (1996) also developed a colorimetric ELISA based on a dual monoclonal antibody pair. Although this ELISA was able to detect elevated VEGF levels in cancer patients, it lacked the sensitivity needed to measure endogenous levels of VEGF in normal individuals. Rodriguez et al. J. Immunol. Methods 219:45 (1998) described a two-site fluorimetric VEGF ELISA that yields a sensitivity of 10 pg/ml VEGF in neat plasma or serum. However, this fluorimetric assay can only detect fully intact 165/165 and 165/110 species of VEGF.
Thus, there is a need to develop a diagnostic and prognostic assay that detects higher measurable levels of VEGF in a biological sample of an animal model or patient than existing ELISAs, and can measure all the isoforms of VEGF.